Previous research has demonstrated a strong genetic component in the development of alcohol abuse. Specifically, people with a high resistance to intoxication at the time of their first drink are at risk. This suggests protein targets of alcoholor their downstream effectors that mediate behavioral intoxication are critical for the potential of a individual to abuse alcohol. A highly conserved target protein that is emerging as a key mediator for behavioral intoxication and tolerance is the big conductance potassium (BK) channel. The BK channel is activated by low levels of alcohol (~20 mM) across many species including C. elegans, rodent and human, which is equivalent to the legal level of intoxication. In mice, this channel contributes to both behavioral intoxication and tolerance. A gain-of function mutation in the BK channel results in hypersensitivity to alcohol in humans. In addition, a genetic screen performed in the model organism C. elegans discovered that null mutations in the worm ortholog of the BK channel, SLO-1, produced an extreme level of resistance to behavioral intoxication. In order to elucidate the interaction of ethanol and the BK channel at the molecular level, we are using two genetic approaches with C. elegans to uncover novel non-null mutations in the worm and human BK channel gene that result in resistance to behavioral intoxication. We will be able to study the human BK channel in the worm because we have humanized the worm by rescuing ethanol sensitivity in a slo-1(null) C. elegans with the human BK channel. First, we will perform a specialized genetic screen to isolate novel non-null mutations in the worm version of BK channel that result in behavioral resistance to intoxication. Second, we will carry out targeted random mutagenesis on the human BK channel gene, and transform the mutated gene into slo-1(null) C. elegans. Transformed C. elegans will be tested for behavioral resistance to intoxication. For both approaches, non-null candidate mutations will identify key portion(s) of the gene that are critical for behavioral intoxication. We will distinguish null from non-null mutations by analysis of locomotory posture and subsequent DNA sequencing. So far, one of the 22 mutants obtained from the first approach has been identified as a candidate non-null mutant. After non-null mutants are identified, we will perform in vivo patch-clamp recordings to assess how the mutation alters basal BK channel function and response to alcohol at the level of single-channel activity. This study will provide knowledge on what residues are critical for ethanol modulation of the BK channel that results in behavioral intoxication across species, and a better understanding of how ethanol interacts with ion channels at the molecular level. PUBLIC HEALTH RELEVANCE: The big conductance potassium (BK) channel is critical for behavioral intoxication and/or tolerance across many species. The goal of this study is to investigate what residues on the BK channel are critical for ethanol modulation at the molecular level, and behavioral intoxication. Results from this study will lead to a better understanding of how ethanol interacts with ion channels at the molecular level, and have implications for treating alcohol abuse.